RNA interference (RNAi) is the process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA. Although RNAi was first discovered in Caenorhabditis elegans (Fire et al., 1998), similar phenomena had been reported in plants (post-transcriptional gene silencing [PTGS]) and in Neurospora crassa (quelling) (reviewed in Hammond et al., 2001; Sharp, 2001). It has become clear that dsRNA-induced silencing phenomena are present in evolutionarily diverse organisms, e.g., nematodes, plants, fungi and trypanosomes (Bass, 2000; Cogoni and Macino, 2000; Fire et al., 1998; Hammond et al., 2001; Ketting and Plasterk, 2000; Matzke et al., 2001; Sharp, 2001; Sijen and Kooter, 2000; Tuschl, 2001; Waterhouse et al., 2001). Biochemical studies in Drosophila embryo lysates and S2 cell extracts have begun to unravel the mechanisms by which RNAi works (Bernstein et al., 2001; Tuschl et al., 1999; Zamore et al., 2000).
RNAi is initiated by an ATP-dependent, processive cleavage of dsRNA into 21- to 23-nucleotide (nt) short interfering RNAs (siRNAs) (Bernstein et al., 2001; Hamilton and Baulcombe, 1999; Hammond et al., 2000; Zamore et al., 2000) by the enzyme Dicer, a member of the RNase III family of dsRNA-specific endonucleases (Bernstein et al., 2001). These native siRNA duplexes containing 5′ phosphate and 3′ hydroxyl termini are then incorporated into a protein complex called RNA-induced silencing complex (RISC) (Hammond et al., 2000). ATP-dependent unwinding of the siRNA duplex generates an active complex, RISC* (the asterisk indicates the active conformation of the complex) (Nykanen et al., 2001). Guided by the antisense strand of siRNA, RISC* recognizes and cleaves the corresponding mRNA (Elbashir et al., 2001b; Hammond et al., 2000; Nykanen et al., 2001).
Recently, Tuschl and colleagues (Elbashir et al., 2001a) have demonstrated that RNAi can be induced in numerous mammalian cell lines by introducing synthetic 21-nt siRNAs. By virtue of their small size, these siRNAs avoid provoking an interferon response that activates the protein kinase PKR (Stark et al., 1998). Functional anatomy studies of synthetic siRNA in Drosophila cell lysates have demonstrated that each siRNA duplex cleaves its target RNA at a single site (Elbashir et al., 2001c). The 5′ end of the guide siRNA sets the ruler for defining the position of target RNA cleavage (Elbashir et al., 2001c). 5′ phosphorylation of the antisense strand is required for effective RNA interference in vitro (Nykanen et al., 2001). Mutation studies have shown that a single mutation within the center of an siRNA duplex discriminates between mismatched targets (Elbashir et al., 2001c). These experiments showed a more stringent requirement for the antisense strand of the trigger dsRNA as compared to the sense strand (Grishok et al., 2000; Parrish et al., 2000). Notably these phenomena were demonstrated in vitro or in cell culture systems.
There is a need for further study of such systems. Moreover, there exists a need for the development of reagents suitable for use in vivo, in particular for use in developing human therapeutics.